In the event of a radiation disaster or attack, high dose radiation exposure to humans causes Acute Radiation Syndrome (ARS). The hematopoietic system and gastrointestinal (GI) tract are among the most vulnerable tissues to radiation injury due to the presence of cycling stem cells that are sensitive to radiation. Loss of regenerative capacity in these organs leads to lethal toxicity. For example, damage to the bone marrow from total body irradiation leads to the hematopoietic syndrome. High doses of radiation to the abdomen results in GI syndrome characterized by damage to the intestinal epithelium, loss of mucosal barrier, and sepsis. While, medical countermeasures to mitigate the hematopoietic syndrome are available, there are currently no FDA approved therapies to mitigate the GI syndrome. We have demonstrated that p53 mediated expression of the cell cycle inhibitor p21 in GI epithelial cells is critical for survival of the GI syndrome. High p21 expression prevents damaged cells from progressing through mitosis and succumbing to mitotic catastrophe. In contrast, radiation kills p21-low, fast cycling LGR5+ GI stem cells in the base of the crypts. When these cells are lost, regeneration of the intestinal epithelium is dependent on a slow-cycling, and thus radioresistant, cell population that resides higher in the crypt. Therefore, identifying the GI epithelial cell type that is preserved by high p21 expression and capable of supporting tissue regeneration following injury will be important for developing mitigation strategies for GI syndrome. Recently published single cell RNA sequencing data from irradiated small intestine epithelial cells in mice identified Clu+ revival stem cells as slow cycling cells with high p21 levels that survive 24 hours and proliferate after high dose irradiation. Clu+ cells are capable of regenerating LGR5+ stem cells after injury and loss of this cell type sensitizes mice to GI syndrome lethality. Clu+ cells rely on the Yap1/Hippo signaling axis for maintenance and therefore we hypothesize that modulation of this pathway will mitigate the GI syndrome. In addition, Dclk1+ tuft cells were also identified to have high p21 expression and survive high dose radiation. These cells are chemosensory and secretory cells that support renewal of the GI epithelium following irradiation by maintaining the stem cell niche. We hypothesize that p53 regulation of p21 in these cell types is critical to intestinal recovery following radiation injury and that targeting these cells for mitigation is a promising strategy. We propose to dissect the mechanisms by which Clu+ revival stem cells and Dclk1+ tuft cells function to promote renewal of the GI epithelium after radiation injury using sophisticated genetically engineered mouse models. We will use targeted approaches with small molecule drugs in vivo and CRISPR/Cas9 screening methods in organoid cultures to identify novel mitigator targets that function specifically in these cell types to reduce lethal GI toxicity.